Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4732—Casein
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/19—Dairy proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
  • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
  • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

• the present invention relates generally to inhibiting the attachment of HaemophHus infiuenzae to human cells, and more specifically to the use of native or recombinant human /.-casein and hydrolysates thereof for inhibiting the attachment of HaemophHus infiuenzae (H. infiuenzae) to human cells.
• Haemophilus are small, gram-negative, non-molotile, non-spore forming bacilli with complex growth requirements. Diseases caused by H. infiuenzae usually begin as a nasopharyngitis, possibly precipitated by a viral infection of the upper respiratory tract. Morse, et al . "Haemophilus", MICROBIOLOGY. FOURTH EDITION, published by J.B. Lipincott Company, pages 615-618 (1990).
• H. infiuenzae are spread from person to person by airborne respiratory droplets or direct contact with secretions. To colonize, H. infiuenzae must contend with ciliary clearance mechanisms of the nasopharyngeal mucosal surface and the mucous barrier. Once past the mucous barrier and the ciliary escalator, H. infiuenzae attach to mucosal epithelial cells. Invasion of mucosal surfaces appears to be an important characteristic of pathogenic bacteria. Stephens, et al . , "Pathogenic Events During Infection of the Human Nasopharynx with Neisseria meningitis and Haemophi lus infiuenzae" .
• H. infiuenzae harbored in the nasopharynx are a key factor in the development of middle ear infections (otitis media), and that non-typable H. infiuenzae adhere to nasopharyngeal and nasal mucosal cells.
• Harada et al . "Adherence of Haemophi lus infiuenzae to nasal, nasopharyngeal and bucal epithelial cells from patients with otitis media" EUROPEAN ARCHIVES OF 0T0-RHINO-LARYNGOLOGY.
• WO 91/06308 filed by Andersson et al . for “ANTIBACTERIAL COMPOSITION", and a published article by the same authors (Aniansson et al., "Anti- adhesive activity of human casein against Streptococcus pneumonia and Haemophilus infiuenzae".
• MICROBIAL PATHOGENESIS. 8:315-323 (1990) disclose the use of a milk fraction having a molecular weight of at least 5,000 daltons for "therapeutic prophylactic, and/or diagnostic use in infections caused by S. pneumonae and/or H. infiuenzae", but it is suggested in these publications that the beneficial effect is provided by kappa-casein.
• the present invention relates to the use of native or recombinant human /.-casein and hydrolysates of both to inhibit H. infiuenzae infections.
• W093/04172 relates to a DNA sequence encoding human /.-casein, but does not disclose the capacity of either native or recombinant human /.-casein to inhibit the attachment of H. infiuenzae to human cells.
• W091/08675 discloses an infant formula which contains recombinant forms of both human alpha-lactalbumin and human /.-casein.
• this publication discloses only that these human milk proteins will "give a simulated human mother ' s milk formula that does not exhibit the allergenic properties associated with formulas based on cow or other foreign protein.” (page 3, lines 20-22).
• the use of human -casein to inhibit the attachment of H. infiuenzae to human cells is not taught or suggested in said publication.
• the two assays (a radiolabeled assay and an ELISA assay) which were used for determining the bioactivity of /.-casein are described below. These assays have not been published heretofore, although the ELISA assay was based upon established methodology.
• Haemophi lus infiuenzae (H. infiuenzae) cultures (fimbriated, nontypable) which have been implicated in otitis media were obtained from Dr. Lauren Bakeletz of The Ohio State University, Columbus, Ohio, U.S.A. The use of these organisms in assays has been described in Bakaletz, et al . , "Frequency of Fimbriation of Nontypable Haemophi lus infiuenzae and its Ability to Adhere to Chinchilla and Human Respiratory Epithelium", Infection and Immunity, 56: 331-335 (1988). The H.
• infiuenzae were streaked onto Chocolate agar plates (BBL-Becton Dickinson & Co., Cockeysville, Maryland, U.S.A.) from frozen aliquots of a low passage number and incubated at 37°C in a 5% C0 2 incubator for about 18 hours to obtain logrith ically growing cultures.
• the H. infiuenzae was used in both the Enzyme Linked Immuno Sorbent Assay (ELISA) assay and the radiolabeling assay as described below.
• ELISA Enzyme Linked Immuno Sorbent Assay
• Human ⁇ -casein cDNA was isolated by Hansson et al. as a 1.1-kb fcoRI fragment from a human lambda gt mammary gland library, and was subcloned into pUC19, which was designated pS21.
• the cDNA was modified by introduction of synthetic oligonucleotides in the 5 ' and 3 ' termini. To introduce a suitable cloning site in the 5 ' end, Ndel . a translational start, was inserted in front of the sequence encoding mature human ⁇ -casein. To adapt the initial part of the translated sequence to E.coli codon usage, six synthetic oligonucleotides were constructed and ligated.
• a 303-bp _ccI/ ⁇ g7II fragment was isolated and cloned into a pUC18 derivative and designated plasmid pS22.
• Four synthetic oligonucleotides containing the sequence encoding the carboxy-ter inal end and translation stop followed by Bam I and EcoRI sites were constructed resulting in the sequence 5 ' AGATCTACCCTGTGA CTCAGCCACTTGCCCCAG ⁇ CATAACCCCA ⁇ AGTGTCTAATAAGGATCCGAA ⁇ C-3' , (SEQ ID NO: 3:) where the protein encoding sequence is underlined.
• the synthetic fragment was cloned into Bglll/EcoRl digested pS22, resulting in plasmid pS23.
• three fragments were ligated: an 89-bp Pstl/Avall fragment from pS24; a 197-bp Avail /Accl fragment from pS21; and Pstl/AccI digested pS23.
• the resulting plasmid pS25 was digested with Nde ⁇ /BamHI and a 641-bp fragment was isolated and cloned into the vector pET-3a.
• the resulting expression vector was designated pS26.
• the E. coli signal sequence of the enterotoxin STII gene was introduced in front of the .-casein encoding sequence.
• a modified STII sequence with Ncol- and Ndel-compatible ends and an internal Clal site was obtained by using a synthetic oligonucleotide, 5 ' -CATGAAAAAGAATATCGCATTTCTTCTTGCATCGATGTTCGTTT TTTCTATTGCTACAAATGCATATG-3' (SEQ ID NO: 4:).
• pS25 was digested with Aval/EcoRI and a 619-bp fragment was isolated.
• This fragment was ligated wi th a syntheti c o 1 i g o n u c 1 e o t i d e fragment , 5'CATATGCACGTGAAACCATCGAATCCCTGAGCTCGAG-3 ' (SEQ ID NO: 5:), and Ndel/EcoRI- digested pUC19.
• the resulting plasmid was designated pS27.
• the final expression vector,pS28 was constructed by ligating three fragments: a 700- bp Ndel/Hindlll ⁇ -casein fragment isolated from pS27, the STII signal sequence, and a Ncol/ Hindi 11-digested pACAT7 vector.
• the expression vectors pS26 and pS28 were used to transform E.coli strains BL2KDE3), BL21(DE3)pLysS, and BL21(DE3)pLysE.
• the bacteria were grown in Luria Broth medium containing 50 ⁇ g/ml carbenicillin, and when B121(DE3)pLysS and BL21(DE3)pLysE were used the medium was supplemented with 25 ⁇ g/ml chloramphenicol .
• the cultures were grown to a density yielding an optical density (OD) of 0.6 at a wavelength of 600 nanometers (OD 600 ), then 0.4 mM IPTG was added to induce the T7 system.
• the cells were harvested about 90 minutes after induction.
• Recombinant /.-casein was isolated using standard procedures.
• the inducible T7-based expression system resulted in high-level expression of recombinant ⁇ -casein.
• Bacteria were harvested and the cells pelletted by centrifugation. The supernatant contained the periplasmic proteins and the pellet the cytoplasmic fraction.
• the recombinant proteins obtained were compared with native ⁇ -casein, which had been purified by standard methods including either ion-exchangechromatography followed by reversed-phaseHPLC or gel filtration.
• Recombinant and native /.-casein were compared by standard biochemical techniques comprising SDS-PAGE, Western blotting, amino acid analysis,peptide mapping, phosphate analysis, and mass spectrometry.
• Recombinant -casein expressed in E.coli was found to comigrate with full- length, nonphosphorylated native human /.-casein, which is one of seven native isoforms.
• Recombinant human /.-casein has also been expressed in S.cerevisiae using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA), but the expression level was approximately 10% of that obtained in E.coli .
• Hansson et al . found that S. cerevisiae appeared to express phosphorylated human milk ⁇ -casein.
• the human /.-casein (both native and recombinant) was digested using the specific endoproteinase GLU-C (Sigma, sequencing grade) which catalyzes the hydrolysis of peptide bonds at the C-terminal of glutamic acid residue. After monitoring the digest using high pressure liquid chromatography, an enzyme to protein ratio of 1:100 (weight/weight) was chosen for a 30 hour digestion at 37°C in 0.1 M NH 4 HC0 3 , pH 7.8. These digests were dried and resuspended in appropriate buffers prior to use in the assays discussed above.
• Detroit 562 pharyngeal carcinoma cells were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.). The use of this type of cell in assays has been described in Takahashi , et al . "Phosphorylation Of A Surface Receptor Bound Urokinase-Type Plasminogen Activator In a Human Metastatic Carcinomatous Cell Line", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 182:1466-72.
• the Detroit 562 cells were cultured in Dulbecco's Modified Eagle Medium (GIBCO, Grand Island, New York, U.S.A.) supplemented with 10% Fetal Bovine Serum (Hyclone, Logan, Utah.
• HBS Hanks Balanced salt solution
• GEBCO Hanks Balanced salt solution
• HBS N-2- Hydroxyethylpeperazine-N'-2-Ethane Sulfonic Acid
• the radiolabeled bacteria were then washed two times and resuspended in 5 ml HBS supplemented with 30 mM HEPES buffer. 25 ⁇ l of the m -In labeled bacterial suspension were preincubated with 25 ⁇ l of ⁇ -casein in a polypropylene 96 well plate for 15 minutes at 37°C to allow binding of /.-casein to bacteria.
• human and bovine -casein solutions were prepared first in 20 mM ethanolamine, 6 M urea, pH 9.5 and then washed twice in PBS by ultrafiltration using Centricon membrane filters (Amicon, MA) with a cut-off of 3,000 daltons. After resuspending in appropriate buffer for the radiolabeled assay described above, these samples were tested in the assay. Experiments with different designated numbers were performed in different days. As shown in Table 1, human /.-casein exhibited an inhibition of 50% or more at concentrations of 0.75 mg/ml or greater.
• Hydrolysate of human /.-casein obtained with GLU-C enzyme was also active (>50% inhiDition) at concentrations of 0.75 mg/ml or higher.
• GLU-C hydrolysate of purified recombinant ⁇ - casein was tested at 3.0 mg/ml , it exhibited activity similar to that of the human milk -casein hydrolysate.
• this recombinant protein could be produced in large-scale from bacteria to provide an abundant supply of a protein which retains the anti-adhesion activity of native human milk ⁇ - casein against H. infiuenzae.
• Oropharyngeal (OP) cells were collected from donors and pooled in phosphate buffered saline (PBS). Cell suspensions washed one time in PBS were resuspended and counted using a hemocytometer. Cells were adjusted to a density of 2.5 x 10 5 cells per ml in PBS. To promote OP cell attachment, 96-well plates (Linbro-ICN, Costa Mesa, California, U.S.A.) were coated with L-lysine followed by exposure to 1.25% glutaraldehyde (creates cross-linking). Plates were thoroughly washed to remove any residual glutaraldehyde.
• PBS phosphate buffered saline
• Each well of the 96-well plate was inoculated with 50 ⁇ l of the OP cell preparation yielding a final concentration of 1.25 x 10 4 cells per well .
• Designated wells were incubated with PBS only (no OP cells) to serve as background control wells to measure non specific bacterial binding to plastic.
• Inoculated plates were centrifuged at 2,700-3,000 rpm for ten minutes to sediment the suspended cell preparation, aspirated and incubated overnight at 37°C in a moist chamber.
• the next morning plates with OP cells were treated with PBS containing 5% BSA for four hours to prevent non-specific bacterial attachment.
• the plates were washed three times with 200 ⁇ l PBS-BSA before use in adhesion assays.
• biotinylated H. infiuenzae were diluted 1:1 with ⁇ -casein or control buffer (PBS-BSA) and incubated at 37°C in a shaking water bath for 30 minutes. 50 ⁇ l of the preincubation mixture was placed into the appropriate well of the OP cell assay plate and incubated for 60 minutes at 37°C. The assay was halted by washing the assay plates three times with PBS-BSA and the plate heat fixed at 65°C for 10 minutes. After the plate cooled to room temperature, 100 ⁇ l of Extravidin-peroxidase conjugate (Sigma), diluted 100-fold in PBS-BSA, was added to each well and the plate incubated for 40 minutes at 37°C.
• Extravidin-peroxidase conjugate Sigma
• This conjugate binds to the biotin- labeled bacteria. Excess unbound conjugate was removed by washing the assay plate three times with PBS-BSA and 100 ⁇ l of peroxidate substrate 2,2 ' - Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) was added to each well. Plates were incubated for 10 minutes and subsequently monitored for color development on a Thermomax 96 well plate reader (Molecular Devices, Menlo Park, California, U.S.A.) until the positive control wells containing OP cells and biotinylated bacteria (no /.-casein) reached an OD 600 of 2.5 to 3.0. Binding results were calculated by averaging the results of three replicates.
• an enteral liquid nutritional product such as infant formula, comprising one or more proteins not contained in human milk in combination with a therapeutically effective amount of at least one of the forms of human ⁇ -casein described in the preceding paragraph.
• an enteral liquid nutritional product such as infant formula
• the attachment of H. infiuenzae to human oropharyngeal cells may be inhibited by administering via a nasal passageway, or as a throat spray, a formulation containing a therapeutically effective amount of at least one of the forms of human -casein identified in the preceding paragraph.
• a nasally administered formulation may be in the form of either drops or a spray.
• enteral, throat spray and nasal products and methods are believed to be effective in inhibiting the attachment of H. infiuenzae to human cells because the interaction of the human /.-casein and H. infiuenzae is believed to occur in the nasopharynx via direct contact rather than following digestion and absorption of the /.-casein.
• human /.-casein may be incorporated into any standard or specialized enteral liquid nutritional product containing at least one protein not found in human milk, such as bovine milk based or soy based infant formulas, and other beverages consumed by young children.
• enteral liquid nutritional product containing at least one protein not found in human milk, such as bovine milk based or soy based infant formulas, and other beverages consumed by young children.
• no proteins or hydrolysates thereof found in human milk, other than ⁇ -casein are contained in the liquid enteral nutritional product.
• Such a product has utility in the treatment and prevention of otitis media in human infants.
• MOLECULE TYPE Cloned cDNA representing the product of a human genomic DNA segment
• nucleotide SEQ ID NO: 1 is the human milk protein, ⁇ -casein.
• GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT 90
• MOLECULE TYPE Synthetic ohgonucleotide
• MOLECULE TYPE Synthetic ohgonucleotide

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• 1995
  • 1995-04-18 WO PCT/US1995/003789 patent/WO1995032728A1/en not_active Application Discontinuation
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